Designing Quantum Capacitive Peptide Interfaces for Electroanalytical Applications.

Redox-active moieties assembled on metallic interfaces have been shown to follow quantum mechanical rules, where the quantum capacitance of the interface (directly associated with the electronic structure of the redox-active moieties) plays a key role in the electron transfer dynamics of the interface. Modifying these interfaces with biological receptors has significant advantages (simplifying molecular diagnostics methods, reducing size, time, and cost while maintaining high sensitivity), enabling the fabrication of miniaturized electroanalytical devices that can compete with traditional ELISA and RT-PCR benchtop assay methods. Owing to their intrinsic characteristics, the use of peptide-based redox-active moieties is a promising chemical route for modifying metallic surfaces, resulting in a high quantum capacitive signal sensitivity. In the present work, different ferrocene-tagged peptides with a structure of Fc-Glu-XX-XX-Cys-NH2 (XX = serine, phenylalanine, glycine) were used to form self-assembled monolayers on gold. The feasibility of using these interfaces in an electroanalytical assay was verified by detecting the NS1 DENV (Dengue Virus) biomarker to compare the efficiency of peptide structures for biosensing purposes. Parameters such as the formal potential of the interface, normalized electronic density of states (DOS), quantum capacitance, and electron transfer rate constants were obtained for Ser-, Phe-, and Gly-peptides. The Gly-peptide structure presented the highest analytical performance for sensing NS1 with a sensitivity of 5.6% per decade and the lowest LOD (1.4 ng mL-1) and LOQ (2.6 ng mL-1), followed by Phe-peptide, whereas Ser-peptide had the lowest performance. This work demonstrates that the use of peptides to fabricate a self-assembled monolayer as a biosensor component has advantages for low-cost point-of-care diagnostics. It also shows that the performance of the sensing interface depends strongly on how the chemistry of the surface is designed as a whole, not only on the redox-active group.

Reagentless Quantum-Rate-Based Electrochemical Signal of Graphene for Detecting SARS-CoV-2 Infection Using Nasal Swab Specimens.

The quantum-rate model predicts a rate k as a frequency for transporting electrons within molecular structures, which is governed by the ratio between the quantum of conductance G and capacitance Cq, such that k = G/Cq. This frequency, as measured in a single-layer graphene appropriately modified with suitable biological receptors, can be applied as a transducer signal that ranges sensitivities within the attomole for biosensing applications. Here, we applied this label-free and reagentless biosensing transducer signal methodology for the qualitative diagnosis of COVID-19 infections, where this assay methodology was shown to be similar to the gold-standard real-time polymerase chain reaction. The quantum-rate strategy for the diagnosis of COVID-19 was performed by combining the response of the interface for detecting the S and N proteins of SARS-CoV-2 virus as accessed from nasopharyngeal/oropharyngeal patient samples with 80% of sensitivity and 77% of specificity. As a label-free and reagentless biosensing platform, the methodology is decidedly useful for point-of-care and internet-of-things biological assaying technologies, not only because of its real-time ability to measure infections but also because of the capability for miniaturization inherent in reagentless electrochemical methods. This approach effectively permits the rapid development of biological assays for surveillance and control of endemics and pandemics.

Sensing the quantized reactivity of graphene.

We demonstrated that the variations measured in the quantum capacitance of single-layer graphene, envisioned here as a conceptual molecular model, depend on the chemical reactivity of the molecule and can be used as an analytical and sensing tool for environmental conditions. The variations are quantized as a function of the environmental changes and can be correlated with chemical reactivity indexes such as chemical hardness and softness. This not only constitutes a proof-of-principle that the chemical reactivity of graphene, as a single molecule, can be determined in situ by measuring the quantum capacitance, but also that these measurements can be used as an analytical tool.

Introducing polymer conductance in diagnostically relevant transduction.

In this work we demonstrate that an impedance derived capacitance method is able to cleanly resolve the resonant conductance characteristics of an electrode-confined polymer film. In decorating the film with receptors, this conductance is thereafter modulated by the capturing of specific targets, demonstrated herein with C-reactive protein. This entirely reagentless and single step marker quantification is relevant to the drive of moving assays to a scaleable format requiring minimal user intervention.

Label-free capacitive assaying of biomarkers for molecular diagnostics.

The label-free analysis of biomarkers offers important advantages in developing point-of-care (PoC) biosensors. In contrast to label-based methodologies, such as ELISA, label-free analysis enables direct detection of targets without additional steps and labeled reagents. Nonetheless, label-free approaches require high sensitivity to detect the intrinsic features of a biomarker and low levels of nonspecific signals. Electrochemical capacitance, [Formula: see text], is a feature of electroactive nanoscale films that can be measured using electrochemical impedance spectroscopy. [Formula: see text] is promising as an electrochemical transducing signal for the development of high-sensitivity, reagentless and label-free molecular diagnostic assays. We used a proprietary ferrocene (Fc)-tagged peptide that is able to self-assemble onto gold electrodes (thicknesses <2 nm) to which any biological receptor can be coupled. When coupled with biological receptors (e.g., a monoclonal antibody), [Formula: see text] exhibited by the redox-tagged peptide changes as a function of the target concentration. We provide herein the steps for the qualitative and quantitative detection of dengue non-structural protein 1 (NS1) biomarker. Detection of NS1 can be used to diagnose dengue virus infection, which causes epidemics each year in tropical and subtropical regions of the world. Including the pre-treatment of the electrode surface, the analysis takes ~25 h. This time can be reduced to minutes if the electrode surface is fabricated separately, demonstrating that [Formula: see text] is promising for PoC applications. We hope this protocol will serve as a reference point for researchers and companies that intend to further develop capacitive devices for molecular diagnostic assays.

Charge transport and energy storage at the molecular scale: from nanoelectronics to electrochemical sensing.

This tutorial review considers how the fundamental quantized properties associated with charge transport and storage, particularly in molecular films, are linked in a manner that spans nanoscale electronics, electrochemistry, redox switching, and derived nanoscale sensing. Through this analysis, and by considering the basic principles of chemical reactivity, we show that 'dry' electronic and 'wet' electrochemical characteristics align within a generalized theoretical capacitative framework that connects charge conductance and electron transfer rate. Finally, we discuss the application of these joint theoretical concepts to key developments in nanosensors.

Introducing mesoscopic charge transfer rates into molecular electronics.

It has been demonstrated that mesoscopic rates operate in nanoscale electrochemical systems and, from a fundamental point of view, are able to establish a bridge between electrochemical and molecular electronic concepts. In the present work we offer additional experimental evidence in support of this statement.

The nanoscopic principles of capacitive ion sensing interfaces.

Herein we discuss the operational principles of molecular interfaces that specifically recruit ions from an electrolyte solution and report this in a reagentless capacitive manner. At low ionic occupancy the response of the interface obeys a Debye-type phenomenon akin to classic "image charge" effects. At higher levels of occupancy, the response follows Thomas-Fermi screening and, significantly, is dependent on the electronic structure of the mesoscopic ion-receptor host-guest ensemble.

Serological point-of-care and label-free capacitive diagnosis of dengue virus infection.

Dengue non-structural protein 1 (NS1 DENV) is considered a biomarker for dengue fever in an early stage. A sensitive and rapid assay for distinguishing positive from negative dengue infection samples is imperative for epidemic control and public health in tropical regions because it enables the development of instantaneous updatable databases and effective surveillance systems. Presently, we successfully report, for the first time, the use of the electrochemical capacitive method for the detection of NS1 DENV biomarker in human serum samples. By using a ferrocene-tagged peptide modified surface containing anti-NS1 as the receptor, it was possible to differentiate positive from negative samples with a p < 0.01 in a reagentless and label-free capacitive format. This capacitive assay had a cut-off of 1.36% (confidence interval of 99.99%); it therefore opens new avenues for developing miniature label-free electrochemical devices for infectious diseases.

Perspectives on and Precautions for the Uses of Electric Spectroscopic Methods in Label-free Biosensing Applications.

Label-free approaches for molecular diagnostic applications are appealing because of their inherent point-of-care advantages. Nonetheless, technical challenges impose a limit on the use of these methods as will be discussed in this paper. Electrochemical spectroscopic methods, such as impedance and impedance-derived methods, are highly effective in the development of label-free diagnostic assays, but they require careful control of the dynamics of the sensing interface. We herein report the strength and challenges of the current methodologies associated with the applications of impedance and impedance-derived methods by focusing on their principles of operation. We demonstrate that the uses of their potentialities are not based on the know-how of these methods, but on how to combine the spectroscopic features with the required chemical design for the associated sensing interfaces. Predominantly, we illustrate how to use the resistive and capacitive terms of the interface to improve its sensitivity to the target. For instance, with the proper signal amplification strategy, limitations related to target-to-receptor size ratio can be overcome. The target-to-receptor ratio is one of the difficulties that we use as an example to illustrate how the sensing of an electric signal can be improved by controlling the properties of the interface on the nanometer scale.

Field effect in molecule-gated switches and the role of target-to-receptor size ratio in biosensor sensitivity.

We demonstrated here that molecular redox films are electrochemical capacitive devices possessing specific field effect in which molecular moieties within films act as sensitive gates. We confirm that the field effect present in these redox switches is suitable in detecting, in a label-free manner (without needs of redox probe in the biological samples), biomarkers of essential importance for dengue, heart risks and inflammation, Parkinson's disease and tumors. Though the sensitiveness is high, it is governed by Thomas Fermi screening and thus depends on the target-to-receptor size ratio. Thus, we also demonstrated how this target-to-receptor size ratio affects the sensitivity. We concluded that the smaller the biological receptor the greater the sensitivity. Consequently, a larger molecular target associated with a smaller receptor provides a considerable (predictable) improvement of the sensitiveness.

Reagentless Detection of Low-Molecular-Weight Triamterene Using Self-Doped TiO2 Nanotubes.

TiO2 nanotube electrodes were self-doped by electrochemical cathodic polarization, potentially converting Ti4+ into Ti3+, and thereby increasing both the normalized conductance and capacitance of the electrodes. One-hundred (from 19.2 ± 0.1 µF cm-2 to 1.9 ± 0.1 mF cm-2 for SD-TNT) and two-fold (from ∼6.2 to ∼14.4 mS cm-2) concomitant increases in capacitance and conductance, respectively, were achieved in self-doped TiO2 nanotubes; this was compared with the results for their undoped counterparts. The increases in the capacitance and conductance indicate that the Ti3+ states enhance the density of the electronic states; this is attributed to an existing relationship between the conductance and capacitance for nanoscale structures built on macroscopic electrodes. The ratio between the conductance and capacitance was used to detect and quantify, in a reagentless manner, the triamterene (TRT) diuretic by designing an appropriate doping level of TiO2 nanotubes. The sensitivity was improved when using immittance spectroscopy (Patil et al. Anal. Chem. 2015, 87, 944-950; Bedatty Fernandes et al. Anal. Chem. 2015, 87, 12137-12144) (2.4 × 106 % decade-1) compared to cyclic voltammetry (5.8 × 105 % decade-1). Furthermore, a higher linear range from 0.5 to 100 µmol L-1 (5.0 to 100 µmol L-1 for cyclic voltammetry measurements) and a lower limit-of-detection of approximately 0.2 µmol L-1 were achieved by using immittance function methodology (better than the 4.1 µmol L-1 obtained by using cyclic voltammetry).

Common Principles of Molecular Electronics and Nanoscale Electrochemistry.

The merging of nanoscale electronics and electrochemistry can potentially modernize the way electronic devices are currently engineered or constructed. This Feature offers a conceptual discussion of this central topic, with particular focus on the impact that uniting physical and chemical concepts at the nanoscale could have on the future development of electroanalytical devices.

Redox Capacitive Assaying of C-Reactive Protein at a Peptide Supported Aptamer Interface.

Electrochemical immunosensors offer much in the potential translation of a lab based sensing capability to a useful "real world" platform. In previous work we have introduced an impedance-derived electrochemical capacitance spectroscopic analysis as supportive of a reagentless means of reporting on analyte target capture at suitably prepared mixed-component redox-active, antibody-modified interfaces. Herein we directly integrate receptive aptamers into a redox charging peptide support in enabling a label-free low picomolar analytical assay for C-reactive protein with a sensitivity that significantly exceeds that attainable with an analogous antibody interface.

Mapping molecular binding by means of conformational dynamics measurements.

Protein-protein interactions are key in virtually all biological processes. The study of these interactions and the interfaces that mediate them play a key role in the understanding of biological function. In particular, the observation of protein-protein interactions in their dynamic environment is technically difficult. Here two surface analysis techniques, dual polarization interferometry and quartz crystal microbalance with dissipation monitoring, were paired for real-time mapping of the conformational dynamics of protein-protein interactions. Our approach monitors this dynamics in real time and in situ, which is a great advancement within technological platforms for drug discovery. Results agree with the experimental observations of the interaction between the TRIM21α protein and circulating autoantibodies via a bridging bipolar mechanism. This work provides a new chip-based method to monitor conformational dynamics of protein-protein interactions, which is amenable to miniaturized high-throughput determination.

A dual marker label free electrochemical assay for Flavivirus dengue diagnosis.

Dengue is a RNA viral illness of the genus Flavivirus which can cause, depending on the pervasiveness of the infection, hemorrhagic dengue fever or dengue shock syndrome. Herein we present an electrochemical label free approach enabling the rapid sensitive quantification of NS1 and IgG (supporting an ability to distinguish primary and secondary infections). Using a bifunctional SAM containing PEG moieties and a tethered redox thiol, both markers are detectable across clinically relevant levels by label free impedance derived redox capacitance. A subsequent frequency specific immittance function approach enables assaying (within seconds) with no impairment of analytical quality (linearity, sensitivity and variance).

Quantum capacitance as a reagentless molecular sensing element.

The application of nanoscale capacitance as a transduction of molecular recognition relevant to molecular diagnostics is demonstrated. The energy-related signal relates directly to the electron occupation of quantized states present in readily fabricated molecular junctions such as those presented by redox switchable self-assembled molecular monolayers, reduced graphene oxide or redox-active graphene composite films, assembled on standard metallic or micro-fabricated electrodes. Sensor design is thus based on the response of a confined and resolved electronic density of states to target binding and the associated change in interfacial chemical potential. Demonstrated herein with a number of clinically important markers, this represents a new potent and ultrasensitive molecular detection enabling energy transducer principle capable of quantifying, in a single step and reagentless manner, markers within biological fluid.

Mapping the ionic fingerprints of molecular monolayers.

We have previously proposed, and experimentally resolved, an ionic charge relaxation model for redox inactive self-assembled monolayers (SAMs) on metallic electrodes in contact with a liquid electrolyte. Here we analyse, by capacitance spectroscopy, the resistance and capacitance terms presented by a range of thiolated molecular films. Molecular dynamics simulations support a SAM-specific energy barrier to solution-phase ions. Once surmounted, the entrapped ions support a film embedded ionic capacitance and non-faradaic relaxation, which can be assigned as a particular case of general electrochemical capacitance.

Conceptual density functional theory for electron transfer and transport in mesoscopic systems.

Molecular and supramolecular systems are essentially mesoscopic in character. The electron self-exchange, in the case of energy fluctuations, or electron transfer/transport, in the case of the presence of an externally driven electrochemical potential, between mesoscopic sites is energetically driven in such a manner where the electrochemical capacitance (C[small mu, Greek, macron]) is fundamental. Thus, the electron transfer/transport through channels connecting two distinct energetic (ΔE[small mu, Greek, macron]) and spatially separated mesoscopic sites is capacitively modulated. Remarkably, the relationship between the quantum conductance (G) and the standard electrochemical rate constant (kr), which is indispensable to understanding the physical and chemical characteristics governing electron exchange in molecular scale systems, was revealed to be related to C[small mu, Greek, macron], that is, C[small mu, Greek, macron] = G/kr. Accordingly, C[small mu, Greek, macron] is the proportional missing term that controls the electron transfer/transport in mesoscopic systems in a wide-range, and equally it can be understood from first principles density functional quantum mechanical approaches. Indeed the differences in energy between states is calculated (or experimentally accessed) throughout the electrochemical capacitance as ΔE[small mu, Greek, macron] = ß/C[small mu, Greek, macron], and thus constitutes the driving force for G and/or kr, where ß is only a proportional constant that includes the square of the unit electron charge times the square of the number of electron particles interchanged.

Mesoscopic behaviour of multi-layered graphene: the meaning of supercapacitance revisited.

The electronic density of states and its contribution to the capacitance of graphene compounds (oxidized and reduced) were investigated using an electrochemical impedance-derived capacitance spectroscopic approach. It is clearly demonstrated that graphene oxide, which is known to exhibit semiconductor electronic characteristics, has little influence on the magnitude of the measured capacitance. Moreover, when graphene oxide is electrochemically reduced to graphene, the capacitance increases dramatically by about three orders of magnitude (from microfaradays to millifaradays). This increased capacitive effect has been interpreted as being directly associated with the electrochemical non-faradaic (super- or ultracapacitive) characteristics of the interface (i.e. associated with its electroactive area, for instance). The results obtained and interpretation made in this work demonstrate that the magnitude of the measured capacitance is a consequence of an electrochemical capacitive phenomenon (mesoscopic in essence; thus, the associated capacitance is equivalently termed mesoscopic capacitance) that energetically contains, in series, both electrostatic (geometrical) and quantum effects, thus being essentially different from those exclusively related to the amount of existing interfacial sites for ions (i.e. beyond those associated with pure double-layer capacitive effects). Conceptually, it is proposed that the mesoscopic capacitance of reduced graphene can be explained mainly through quantum chemical effects, ultimately following first-principles quantum mechanics supported on density functional theory, wherein the density of states is central.